Eukaryotes have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to proteins, and often these proteins are then targeted for degradation by the proteasome. Substrates include naturally short-lived regulatory factors and aberrant protein quality control (PQC) substrates. Many human disorders, including neurodegenerative diseases such as Alzheimer's and Parkinson's disease, diabetes, cystic fibrosis, and different forms of cancer, are associated with abnormalities in Ub-dependent proteolysis. The Ub-proteasome system presents promising drug targets for treating these diseases. In this renewal, the PI proposes to extend studies on Ub-dependent proteolysis, focusing on endoplasmic reticulum (ER)- associated degradation (ERAD) and basic features of membrane and nuclear protein ubiquitination and degradation. The proposed research will concentrate on the yeast Saccharomyces cerevisiae because of its experimental advantages and the fact that the Ub system in general, and the ERAD machinery in particular, is highly conserved. Recent work has identified a yeast Ub-ligase complex embedded in the ER and nuclear envelope membranes that is capable of recognizing a wide array of regulatory and PQC substrates. This unusual complex includes a large integral membrane Ub ligase (E3) called Doa10 and two Ub-conjugating enzymes (E2s), Ubc6 and Ubc7. Doa10 is the prototype for a broadly conserved class of viral and eukaryotic Ub ligases. It was discovered from an analysis of a soluble nuclear substrate, the Mat2 transcription factor, but it also has membrane substrates. The latter proteins need additional factors that are not required for the degradation of soluble targets. The overarching goal of the proposal is to define the key biochemical, structural and cell biological determinants of specificity for this highly conserved Ub-ligation system. The following Aims are proposed: (1) Determine how Doa10 interacts with its cofactors and recognizes its substrates by point mutagenesis coupled with genetic and biochemical assays; (2) Investigate how Doa10-dependent degradation of integral membrane proteins differs from that of soluble ones using matched soluble and membrane substrates bearing identical degradation signals (degrons); and (3) Compare different Doa10-dependent degrons to determine if common features characterize both naturally short-lived and PQC substrates and examine Doa10- dependent ubiquitination in the context of the native Mat2 protein.